By way of background, it is appropriate first to describe in summary a procedure which has been developed for the synthesis of an important tetracycline precursor from which oxytetracycline, having the structure of formula (5) above, may be readily obtained.
The procedure for the chemical synthesis of a tetracycline precursor from which oxytetracycline may be ultimately obtained is schematically set forth in the drawing.
It will be appreciated that the specific precursor -- compound (20), shown in the drawings -- is also accessible through microbial synthesis coupled with suitable chemical manipulation of the fermentation product. It is also pointed out that the present invention is concerned not only with the cis 12a hydroxylation of this specific precursor but also with the stereospecific or stereoselective hydroxylation of tetracycline precursors generally as defined in formulas 1B and 1D above, however these precursors may be prepared. A wide variety of precursors may be prepared, for example, following the basic procedure illustrated with specific reference to the preparation of oxytetracycline, making suitable substitutions and modification of reactants where other tetracycline compounds are desired.
The tetracycline precursor for the synthesis of oxytetracycline is assembled from three basic building blocks: The thiazolone of the structure, ##SPC6##
methyl 3-oxoglutaramate having the formula ##SPC7##
and the aldehyde having the formula ##SPC8##
The thiazolone (6) is prepared by the treatment of thiobenzoglycine as described, for example, by Muxfeldt et al. in Journal of the American Chemical Society, Volume 89, pp. 4991-4996 (Sept. 13, 1967).
Methyl 3-oxoglutaramate, compound (7), is obtained by acid hydrolysis of the enamine ##SPC9##
The enamine (9) is, in turn, prepared by a carefully controlled treatment of dimethyl-3-oxoglatarate with ammonia in methanol and is characterized by a melting point of 120.degree.-121.degree. C.
The aldehyde (8) is prepared starting with the addition of 1-acetoxybutadiene (11) to juglone acetate (10). [See drawing.] The tricyclic adduct (12) is converted to the aldehyde (13). A procedure for effecting this conversion is set forth in detail in the paper by Muxfeldt appearing in Angewandte Chemie, Volume 74, pp. 825-828 (1962). Briefly summarized, the procedure involves treating the adduct (12) in a Grignard reaction with methyl magnesium iodide which adds a methyl group to the 9a position. ##SPC10##
This intermediate is treated with absolute acetone and anhydrous copper sulfate to obtain the acetonide: ##SPC11##
Oxidation of the acetonide (12b) with potassium chlorate in the presence of a catalytic amount of osmium tetroxide resulted in the diol: ##SPC12##
Conversion of the diol (12c) to the aldehyde (13) was effected in a two-step reaction in which the diol was first oxidized with lead tetraacetate to form the dialdehyde (12d): ##SPC13##
which, in turn, was cyclized to form the aldehyde (13).
Conversion of the aldehyde (13) to the mixed isomeric aldehydes (14) and (15) is performed by ozonolysis of aldehyde (13), water treatment of the resulting ozonide and cleavage of the reaction product with sodium carbonate. The pure isomers (14) and (15) were characterized by melting points of 140.degree.-143.degree. C and 171.degree.-173.degree. C, respectively.
Piperidine, in refluxing benzene, converted the aldehydes (14) and (15) to the enamine (16) in a 91 percent yield. Enamine (16) was characterized by a melting point of 118.degree.-119.degree. C. The enamine was alkylated with chloromethyl methyl ether to the methoxymethyl ether (17) (m.p. 81.degree.-84.degree. C). When (17) was adsorbed on deactivated silica gel, selective hydrolysis of the enamine function occurred, and the oily aldehyde (8) was formed in a 72 percent yield. The nuclear magnetic resonance spectrum was consistent only with a trans coplanar relationship of the hydrogens at the C-5 and C-5(a) positions, indicating that the hydrolysis through which the aldehyde (8) was formed was stereospecific.
Condensation of the aldehyde (8) with thiazolone (6) in the presence of basic lead acetate in tetrahydrofuran results in thiazolone (18) characterized by a melting point of 157.degree.-160.degree. C.
A combination of strong bases (for example, butyllithium and potassium t-butoxide) catalyzed the reaction of thiazolone (18) with methyl 3-oxoglutaramate (7) to yield the tetracyclic compound (19) characterized by a melting point of 225.degree.C (with decomposition). In general, any strong base may be used which does not destroy the desired tetracyclic product. The reaction was carried out under reflux conditions with tetrahydrofuran as a solvent. The methoxymethyl group was then removed by acetic acid to obtain the tetracycline precursor (20).
The present invention is concerned with the treatment of tetracycline precursors of formula (1B) (compound (20) being a typical such precursor) under conditions resulting in the introduction of a hydroxyl group at the 12a position in a predominantly cis position relative to the 4a hydrogen. More specifically, in accordance with the present invention, introduction of this hydroxyl group is achieved by reacting compound (20) with molecular oxygen in a basic medium employing a non-protic solvent. For the best yields, a peroxide-destroying agent is also provided in the reaction system.
The solvent employed in the present invention is one which does not destroy the desired product in the presence of a strong base. In general these are nonprotic solvents, i.e., solvents which do not release protons in the presence of a strong base. Exemplary solvents include, but are not limited to benzene, toluene, xylene, diglyene, tetrahydrofuran, dioxane, diethyl ether, anisole (as well as a variety of other ethers), dimethyl formamide, dimethyl sulfoxide and ethyl acetate. Other solvents analogous to the foregoing will be obvious to those skilled in the art, including some of the solvents mentioned in the above-cited U.S. Pat. No. 3,188,348.
The strong base used in the present invention must be one having sufficient strength to ionize the hydroxy groups in the tetracycline structure, but, at the same time, be one which will not destroy the product. In general, bases such as the alkali metal amides, the alkali metal butoxides and alkyls of 1-6 carbons and hydrides. Sodium hydride, sodamide, lithium alkyl and potassium t-butoxide are typical. It will be apparent that some of these materials, for example potassium hydride, are hazardous to handle, and are preferably avoided where more convenient alternative materials are available. As a rule, the polyvalent alkoxides, hydrides, alkyls, and the like have been found to be unsuitable, although magnesium alkoxides can be used. The alkali metal hydroxides are also unsuitable because of their destructive effect on the tetracycline.
Finally, the presence of a peroxide-destroying agent is preferred since any peroxides formed during the oxygenation step will destroy the desired tetracyclic product and thereby reduce the yield. Suitable peroxide-destroying agents are trialkyl phosphites, palladium or platinum metal, alkali metal ascorbates, peroxidase enzymes, mercaptans, sulfides, sulfones, and various phosphines. The peroxide-destroying agent should be one which will not attack the tetracycline structure itself. Obviously, materials which are difficult to handle such as the mercaptans and sulfides are not preferred.
It has been found that a small amount of moisture is sometimes necessary to initiate the reaction. Where the reaction seems to start with difficulty, a few drops of water may be added to the reaction mixture.
Treatment of a tetracycline precursor, of which compound (20) is representative, under the foregoing conditions with dry molecular oxygen (either pure oxygen or air) for a period of 2 to 15 minutes. The temperature is typically about room temperature, although in principle any temperature between the freezing point and boiling point of the solvent may be used.
In the preferred practice of the present invention the time of the reaction is adjusted so that it is just sufficient to consume the starting material. This may be conveniently accomplished by following the course of the reaction photometrically or colorimetrically. This have been found to optimize the yield. Extended reaction beyond that just sufficient to consume the reaction product tends to cause degradation and loss of the desired product.
Acid treatment of the hydroxylated product removes the acetonide group bridging the C-5 and C-6 positions of the tetracycline ring structure. Conversion of tetracycline (21) into oxytetracycline (5) is then but a simple step involving removal of the thiobenzamide chromophore and substituting a dimethylamino group.
The present invention is more fully illustrated by reference to the following examples: